1. Field of the Invention
The present invention relates to methods for producing heterologous polypeptides in trichothecene-deficient filamentous fungal mutant cells. The present invention also relates to such mutant cells of filamentous fungal cells and methods for obtaining the mutant cells. The present invention also relates to isolated trichodiene synthases and isolated nucleic acid sequences encoding the trichodiene synthases. The present invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing the trichodiene synthases. The present invention further relates to mutants cells comprising a marker-free modification of a gene, and methods for obtaining and using such mutant cells.
2. Description of the Related Art
Trichothecenes are sesquiterpene epoxides which are named after the fungus Trichothecium roseum from which the first trichothecene was isolated. The trichothecenes T-2 toxin, diacetoxyscirpenol, and deoxynivalenol are most commonly found in agricultural commodities infected with Fusarium species. Interest in these compounds is due primarily to the discovery that trichothecene contamination of foods and feeds may be detrimental in humans and animals.
Trichothecenes are produced by a sequence of oxygenations, isomerizations, cyclizations, and esterifications leading from trichodiene, which is produced from the cyclization of trans, trans-farnesyl pyrophosphate by the enzyme trichodiene synthase (Desjardins, Hohn, and McCormick, 1993, Microbiological Reviews 57: 595-604).
The trichodiene synthase gene (tri5 or tox5) has been cloned from Fusarium sporotrichioides (Hohn and Beremand, 1989, Gene 79: 131-138); Gibberella pulicaris (Hohn and Desjardins, 1992, Molecular Plant-Microbe Interactions 5: 249-256); Gibberella zeae (Proctor et al., 1995, Molecular Plant-Microbe Interactions 4: 593-601); Myrothecium roridin (Trapp, et al., 1995, Journal of Cellular Biochemistry Supplement 19B: 154); and Fusarium poae (Fekete et al., 1997, Mycopathologia 138: 91-97).
Tri5 mutants of Gibberella pulicaris (Hohn and Desjardins, 1992, supra) and Gibberella zeae (Proctor et al., 1995, supra) have been generated which do not produce trichothecenes.
Other genes in the trichothecene biosynthetic pathway have been cloned including the tri3 gene, encoding a 15-O-acetyltransferase, from Fusarium sporotrichioides (McCormick et al., 1996, Applied and Environmental Microbiology 62: 353-359); the tri4 gene, encoding a cytochrome P450 monooxygenase, from Fusarium sporotrichioides (Hohn et al., 1995, Molecular and General Genetics 248: 95-102); the tri6 gene, encoding a zinc finger protein involved in the regulation of trichothecene biosynthesis, from Fusarium sporotrichioides (Proctor et al., 1995, Applied and Environmental Microbiology 61: 1923-1930); the trill gene, encoding a cytochrome P450 monooxygenase required for C-15 hydroxylation, from Fusarium sporotrichioides (Alexander et al., 1997, Applied and Environmental Microbiology 64: 221-225); the tri12 gene, which encodes an apparent transport protein involved in trichothecene production, from Fusarium sporotrichioides (Alexander et al., 1997, Cereal Research Communications 25: 347-348); and the tri101 gene, encoding a 3-O-acetyltransferase, from Fusarium graminearum (Kimura et al., 1998, Journal of Biological Chemistry 272: 1654-1661).
It is an object of the present invention to provide methods for producing polypeptides in mutant cells.